aniline-blue and Infertility--Male

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men.. Case-control study.. Basic science laboratory.. Fertile and infertile men.. Not applicable.. The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis.. Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men.. Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Topics: Case-Control Studies; Chromomycin A3; Histones; Humans; Infertility, Male; Male; Meiosis; Protamines; Proteomics; Semen; Sperm Motility; Testis

The aim of this study was to investigate the possibility of using sperm function tests (hypoosmotic swelling test [HOS], aniline blue [AB] staining test, and sperm chromatin dispersion [SCD]) to predict intrauterine insemination [IUI] success rate. A total of 243 couples with mild male factor or unexplained male infertility who underwent IUI were evaluated prospectively. The results of basic sperm analysis and sperm function tests were compared between pregnant or nonpregnant groups. The HOS (11.9 ± 9.6% vs. 10.1 ± 8.5%, p = 0.35) and SCD tests (32.9 ± 21.0% vs. 29.9 ± 19.0%, p = 0.48) were not significantly different between pregnant (n = 22) and nonpregnant (n = 221) groups. However, the AB staining negativity rate was significantly higher in the pregnant group compared to the nonpregnant group (35.2 ± 20.8% and 24.4 ± 18.0%, p = 0.008). On ROC analysis, a cut-off value of 24% for AB negativity showed a sensitivity and a specificity value of 82.35% and 51.38% (AUC) = 0.653; 95% confidence interval: 0.571-0.72 P (Area = 0.5) = 0.0267, respectively, for prediction of pregnancy. Our study showed that the sperm chromatin maturity, assessed by AB stain, may predict the pregnancy in couples with unexplained female infertility plus mild male factor or unexplained male infertility. The HOS and SCD failed to predict the pregnancy in this group of couples.

Topics: Adult; Aniline Compounds; Chromatin; Chromatin Assembly and Disassembly; Female; Humans; Infertility, Female; Infertility, Male; Insemination, Artificial, Homologous; Male; Predictive Value of Tests; Pregnancy; Pregnancy Rate; Prospective Studies; Semen Analysis; Spermatozoa

According to numerous assisted reproductive medicine practitioners, semen with normal characteristics might not require further investigation. However, on the scale of the individual spermatozoon, it is well known that normal morphology does not guarantee optimal nuclear quality. Here, for 20 patients with normal sperm characteristics and a high proportion of spermatozoa with noncondensed chromatin, we subsequently assessed chromatin condensation status (aniline blue staining) and morphology (Papanicolaou staining) of the same 3749 spermatozoa. Although the overall proportion of morphologically normal spermatozoa was not correlated with the overall proportion of spermatozoa with noncondensed chromatin, an individual spermatozoon's morphology appeared to be closely related to its chromatin condensation status. Morphologically normal spermatozoa with noncondensed chromatin were seen in all patients; the proportion averaged 23.3% [min 10.9%-max 44.4%]. Morphologically abnormal spermatozoa were more likely to have noncondensed chromatin than morphologically normal ones (P < 0.0001). Small-, large- or multiple-headed spermatozoa presented the highest degree of noncondensation (>80% for each type), and more than half the vacuolated spermatozoa also presented noncondensed chromatin. However, a morphologically normal spermatozoon may also have a noncondensed chromatin.

Topics: Aniline Compounds; Centrifugation, Density Gradient; Chromatin Assembly and Disassembly; Coloring Agents; Humans; Infertility, Male; Male; Sperm Head; Spermatozoa

To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies.. The two variables were examined by double probing of the same spermatozoa.. Academic Andrology Laboratory.. Semen samples subjected for analyses were examined.. We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes.. Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated.. The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm.. The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.

Topics: Aneuploidy; Aniline Compounds; DNA Damage; Fluorescent Dyes; Histones; Humans; In Situ Hybridization, Fluorescence; Infertility, Male; Karyotyping; Male; Semen Analysis; Spermatozoa; Staining and Labeling

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

Topics: Aniline Compounds; Apoptosis; Caspase 3; Cell Shape; Creatine Kinase; Cytoplasm; DNA Fragmentation; Histones; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Models, Biological; Research Design; Sperm Maturation; Sperm-Ovum Interactions; Spermatozoa; Staining and Labeling

To study whether the results of tests of sperm chromatin and deoxyribonucleic acid (DNA) normality are related to fertilization rates in vitro.. Normal morphology, nuclear maturity determined by acidic aniline blue stain, and DNA normality determined by acridine orange fluorescence of sperm in insemination medium and the number of sperm bound to the zona pellucida (ZP) of the oocytes that had failed to fertilize in vitro were determined. The relationship between sperm test results and fertilization rates were analyzed by logistic regression.. Samples were obtained from patients undergoing in vitro fertilization (IVF) treatment.. The number of sperm bound to the ZP, the percentage of sperm with normal morphology, and the percentage of sperm with normal DNA were the most significant factors related to fertilization rates in vitro. In patients with normal morphology > or = 15% or with > 10 sperm bound per ZP, the percentage of sperm with normal DNA, the number of sperm bound to the ZP, and motility grade were significantly related to IVF rates.. In patients with normal morphology > or = 15%, failure of fertilization may be because of defects of sperm-ZP binding or abnormal DNA. Assessment of DNA normality of motile sperm in the insemination medium may aid prediction of fertilization rates in addition to normal morphology and sperm-ZP binding.

Topics: Acridine Orange; Aniline Compounds; Cell Nucleus; Chromatin; DNA; Female; Fertilization in Vitro; Fluorescent Dyes; Humans; Infertility, Male; Male; Microscopy, Fluorescence; Regression Analysis; Sperm Motility; Spermatozoa; Staining and Labeling; Zona Pellucida

During mammalian spermiogenesis, replacement of the somatic histones by basic proteins, the protamines, allows normal sperm nuclear condensation. In this study we have evaluated the degree of chromatin compaction in spermatozoa from 191 infertile subjects, affected by different testicular disorders, compare with that in 50 fertile sperm donors (controls). In infertile men, there was a higher percentage of unstable spermatozoa after incubation with sodium dodecyl sulphate (SDS) and of stained spermatozoa after staining with aniline blue (P less than 0.001 vs. controls). Furthermore, a positive linear correlation was found between SDS-unstable spermatozoa and stained spermatozoa (P less than 0.001), suggesting that sperm instability was related to a defect in histone-replacement by sperm-specific nucleoproteins, protamines. When the patients were considered according to pathology, high sperm nuclear instability and a high percentage of stained spermatozoa were detected in groups affected by varicocele, idiopathic infertility and in patients with a history of unilateral cryptorchidism. In the latter group the same alterations were observed even when the cryptorchid testis had been removed during surgery. In the group with a past history of mumps orchitis these parameters did not show any difference when compared with controls.

Topics: Adult; Aniline Compounds; Cell Nucleus; Chromatin; Fluorescent Dyes; Histones; Humans; Infertility, Male; Male; Nucleoproteins; Sodium Dodecyl Sulfate; Spermatozoa

Chromatin condensation is vital for the function of the spermatozoon as the motile carrier of the paternal genome. The degree of condensation can be shown with the aid of acidic aniline blue staining, which is able to discriminate between lysine-rich histones and arginine- and cysteine-rich protamines. Using this technique and employing the Düsseldorf classification of sperm morphology in cases of disturbance of spermatogenesis, it was demonstrated that chromatin condensation is impaired not only in malformed but also in morphologically normal spermatozoa. Among morphologically normal spermatozoa, the percentages of spermatozoa with chromatin condensation disturbances increase in patients with different patterns of sperm malformation, if compared with patients with normozoospermia.

Topics: Aniline Compounds; Chromatin; Fertilization in Vitro; Fluorescent Dyes; Humans; Infertility, Male; Insemination, Artificial; Male; Reference Values; Spermatozoa

A retrospective study of 49 men with proven fertility and 396 suspected infertile men was conducted with the primary objective of investigating the relationship between the nuclear maturity of sperm and male fertility. Acidic aniline blue staining was used to detect chromatin defects of sperm nuclei related to their nucleoprotein content as associated with DNA. The discriminant value of the percentage of unstained nuclei (= percentage of mature heads, MH) and of other semen characteristics, was analysed by a stepwise (forward) linear regression model. Semen characteristics that discriminated significantly between the two groups of subjects were, in descending order: (1) the percentage of normal sperm, (2) the percentage of amorphous heads, (3) the percentage of tapered heads, (4) semen volume, and (5) the percentage of MH. The discriminant value of each of the significant characteristics was studied by means of ROC-curves. MH had the best ROC-curve profile; its cut-off value was found to be equal to 70% (74.5 +/- 2.6% for the donor group versus 53.0 +/- 1.1% for the patient group). A simple infertility score (SIS) including MH, was built according to the cut-off values inferred from the ROC-curves. SIS allowed an overall satisfactory separation of the two groups (less than or equal to 4 = fertile, 5-6 = uncertainty zone, greater than 6 = infertile). Our results indicate that the addition of the evaluation of sperm head maturity to routine semen analysis improves the assessment of fertility in men.

Topics: Aniline Compounds; Cell Division; Cell Nucleus; Chromatin; Fluorescent Dyes; Humans; Infertility, Male; Male; Retrospective Studies; Semen; Spermatozoa; Staining and Labeling; Statistics as Topic